The invention relates to the use of early embryonic cells to deliver genetic material into a fully formed animal.
It is often desirable to confer upon an animal a particular genetic trait.
It is possible to remove bone marrow cells from the animal, transform them with a vector carrying the desired gene, and reimplant the transformed cells. Generally speaking, the transformed cells are given a competitive advantage. For example, the animal may be irradiated to partially or completely destroy the normal marrow, thus providing the transformed marrow cells with a vacant ecological niche. See, e.g., Joyner, et al., Nature (London), 305: 556 (1983). Clearly, this damage to the host is undesirable in a practical genetic delivery system.
Salser, U.S. Pat. Nos. 4,396,601 and 4,497,796 removed bone marrow cells from mice, cotransformed them with DNA including HSV DNA and the marker DHFR gene, and selected for cells resistant to methotrexate. These drug-resistant cells were injected into irradiated mice treated with methotrexate. Preferably, the bone marrow cell population used was one rich in hematopoietic stem cells. Salser does not teach use of cells removed from a mammalian embryo, and Salser severely stressed the recipient mice to give the modified cells a selective advantage.
Wagner et al, WO 82/04443 placed exogenous material into the pronucleus of a zygote. They teach that the zygote should be transformed as soon as possible after fertilization. We transform embryonic cells at a considerably later stage of development.
Mintz and Illmensee, PNAS 72:3585 (1975) injected teratocarcinoma (embryonal carcinoma, EC) cells into mouse blastocysts, obtaining mosaic mice. The teratocarcinoma cells proved to be developmentally totipotent, developing into a variety of normal tissues. This method cannot be used to deliver genes into specific tissues because the developmental course of these cells is uncertain. EC-like totipotent cells (EK cells) have also been obtained from culturing ICM cells of normal mouse embryos removed on day 2.5. Evans and Kaufman, Nature, 292:154 (1981).
We have found a convenient method for delivering genes into specific tissues of an animal which does not require extraction of any cells from the animal. Rather, we transform post-gastrular embryonic cells, which, besides being easier to culture, may be selected to be both (1) predestined to develop into the target tissue, and (2) essentially non-immunogenic.
One method known for the transformation of explanted cells involves use of a retroviral vector. See Vande Woude, U.S. Pat. No. 4,405,712.
When a retrovirus infects a cell, its RNA genome acts as a template for the reverse transcription of the viral genetic information into a double strand of DNA. This DNA molecule, now called a provirus, integrates into the genome of the host. Retroviral RNA is synthesized from the proviral sequence by the host's own RNA polymerase, and some of this RNA is translated into viral proteins. Under the instruction of the packaging sequence (called psi in the Moloney murine leukemia virus studies), the RNA-protein core of the virus is packaged into a glycoprotein envelope, and the resulting viral particle buds off from the cell into the medium (where it may find and infect other cells).
Mann, et al., Cell, 33:153 (1983) developed a cell line, known as psi-2, which is a line of NIH 3T3 cells with a permanently integrated helper virus. The helper virus, psi-minus, corresponds to the MoMLV with the psi sequence deleted by BalI-PstI cleavage. The psi-2 cells produce viral particles only when transformed by a retroviral vector bearing the psi sequence.
Cone and Mulligan, PNAS 81:6349 (1984), of the same research group, later developed an improved packaging cell line, psi-AM. This .cell line was developed by transforming NIH 3T3 cells with a psi-minus chimera of an amphotrophic retrovirus (4070A). This amphotrophic murine retrovirus could infect non-murine hosts, including human and monkey cells.
Both psi-2 and psi-AM cells are readily available in the scientific community.
Joyner et al., supra, used an MoMLV retroviral vector to transfer a neomycin resistance gene into mouse hematopoietic progenitor cells. Williams, et al., Nature (London) 310: 476 (1984) used MSV DHFR-NEO transformed psi-2 cells to transfer neomycin resistance to co-cultivated bone marrow cells. See also Greenberger, et al., PNAS, 80:2931 (1983); Dick, et al., Cell, 42:71 (August 1985); Rubinstein, et al., 81:7137 (1984); Rothstein, et al., Blood, 65:744 (1985).
The above references teach retroviral transformation of "primitive but committed" non-embryonic cells. "Primitive" is a relative term, and these hematopoietic bone marrow stem cells are much further advanced in development than are the embryonic cells of the immediate post-gastrular stage ("neodetermined"), and therefore are likely to be less pluripotent and less histocompatible.
Verma, et al. , in Tumor Viruses and Cell Differentiation, 251 (Scolnick and Levine, eds., 1983) and Miller, et al., PNAS (USA) 80: 4709 (1983) and Science, 225: 630 (1984) also describe use of retroviral vectors in gene therapy.
Genes may also be inserted by other techniques, such as calcium phosphate-mediated DNA uptake. Wigler, et al., Cell, 11: 223 (1977). To assure survival and proliferation of the transformed cells, powerful selection systems, such as DHFR/methotrexate, are used to inhibit untransformed cells. Carr, et al., Blood, 62: 180 (1983); Cline, et al., Nature, 284: 422 (1980). Without such selection, the efficiency of this procedure is presently too low to affect the recipient's condition significantly.
While Hammer, et al., Nature (London) 311: 65 (1984) has used microinjection of an RGH gene to correct dwarfism in the mouse, the technique is too labor intensive to be commercially practicable, even if other difficulties were overcome.
Lipid vesicles containing exogenous DNA have been injected into the tail vein of mice, so transformation occurs in vivo. Szoka, U.S. Pat. No. 4,394,448.
Kiester, Jr., Science 86, at 33 (March 1986) reports on research in which rat fetal brain tissue was grafted intraocularly into adult rats.
Jacob, EP Appl. 178,220 used a retroviral vector to confer G418 resistance on three embryonal carcinoma cell lines. He teaches implanting genetically engineered embryos into the uterus of a female mammal where it may naturally develop into a transgenic infant. This is to be distinguished from the present invention, in which engineered embryonic cells are injected into the bloodstream, or the corresponding tissue of the recipient. Jacob also teaches removing bone-marrow cells from a postnatal animal, transforming the cells, and returning them to the same animal.
Heit et al., in The Biology of Bone Marrow Transplantation, 507-517 (1980) suggested that fetal liver cells could be used for hematopoietic reconstitution without a graft versus host reaction. Mouse fetal liver cells have been microinjected into the placental circulation and thereby introduced into a recipient fetus. While this technique permits donor hematopoietic cells to become competitively established without ablation or irradiation of the recipient, it is dependent on the immunological immaturity of both donor cells and recipient. Fleischman, et al., Cell, 30:351-359 (1982); Flake, et al., Science, 233:776-778 (1986). Segal, et al., Transplantation, 28:88-95 (1979) suggests a mechanism whereby fetal bone grafts may escape host rejection in immunocompetent hosts.
Japanese application 61-81743 is said to relate to "a mature, non-human animal containing germ and somatic cells transformed by an activated tumor sequence, which was introduced into the animal or its ancestor during the fetal stage."
Yolk sac cells which produce an embryonic variant hemoglobin have been injected into irradiated adult mice of another strain and the surviving mice were found to be producing, in part, the donor adult type of hemoglobin. Auerbach, in EPITHELIAL-MESENCHYMAL INTERACTIONS, ch. 13 (1968).